Loss of nitrogen (N) from land limits the uptake and storage of atmospheric CO(2) by the biosphere, influencing Earth's climate system and myriads of the global ecological functions and services on which humans rely. Nitrogen can be lost in both dissolved and gaseous phases; however, the partitioning of these vectors remains controversial. Particularly uncertain is whether the bacterial conversion of plant available N to gaseous forms (denitrification) plays a major role in structuring global N supplies in the nonagrarian centers of Earth. Here, we use the isotope composition of N ((15)N/(14)N) to constrain the transfer of this nutrient from the land to the water and atmosphere. We report that the integrated (15)N/(14)N of the natural terrestrial biosphere is elevated with respect to that of atmospheric N inputs. This cannot be explained by preferential loss of (14)N to waterways; rather, it reflects a history of low (15)N/(14)N gaseous N emissions to the atmosphere owing to denitrifying bacteria in the soil. Parameterizing a simple model with global N isotope data, we estimate that soil denitrification (including N(2)) accounts for approximately 1/3 of the total N lost from the unmanaged terrestrial biosphere. Applying this fraction to estimates of N inputs, N(2)O and NO(x) fluxes, we calculate that approximately 28 Tg of N are lost annually via N(2) efflux from the natural soil. These results place isotopic constraints on the widely held belief that denitrifying bacteria account for a significant fraction of the missing N in the global N cycle.